The present invention relates to a novel metalloproteinase useful in applications such as diagnosis of the presence of tumour cells, diagnosis of the degree of tumour malignancy., or other medical or physiological fields.
More specifically, the present invention relates to one type of metalloproteinase expressed specifically in human tumour cells and a DNA sequence encoding therefor; a plasmid having a nucleotide sequence which contains said DNA sequence; a host cell harbouring said plasmid; a method for manufacturing said protein using said host cell; a probe which hybridizes with the aforesaid DNA sequence; a method for detecting DNA or RNA containing the aforesaid sequence using said probe; and monoclonal and polyclonal antibodies which bind specifically to the aforesaid protein.
A group of enzymes with different substrate specificity and referred to in general as matrix metalloproteinases (hereinafter referred to as xe2x80x9cMMPsxe2x80x9d) contributes to degradation of the extracellular matrix comprising such complex components as collagen, proteoglycan, elastin, fibronectin, and laminin.
Previously reported MMPs include interstitial collagenase (MMP-1), 72 kDa gelatinase (also known as type IV collagenase or gelatinase A; MMP-2), 92 kDa gelatinase (also known as type IV collagenase or gelatinase B; MMP-9), stromelysin-1 (MMP-3), matrilysin (MMP-7), neutrophil collagenase (MMP-8), stromelysin-2 (MMP-10) and stromelysin-3 (MMP-11).
These MMPs are a family of enzymes whose primary structure has been reported previously. With the exception of MMP-7, the primary structure among the family of reported MMPs comprises essentially an N-terminal propeptide domain, a Zn+ binding catalytic domain and a C-terminal hemopexin-like domain. In MMP-7 there is no hemopexin-like domain. MMP-2 and MMP-9 contain an additional gelatin-binding domain. In addition, a proline-rich domain highly homologous to a type V collagen xcex12 chain is inserted in MMP-9 between the Zn+ binding catalytic domain and the C-terminal hemopexin-like domain.
In highly metastatic tumour cells, there are reports of conspicuous expression of type Iv coliagenase (MMP-2, MMP-9) which mainly degrade type IV collagen (Cancer Res., 46:1-7, 1986; Biochem. Biophys. Res. Commun., 154:832-838, 1988; Cancer, 71:1368-1383, 1993). Likewise, it has been reported MMP-3 act as an activator of proMMP-9 (J. Biol. Chem., 267:3581-3584, 1992).
The degree of matrix metalloproteinase expression serves as an index to diagnosing the degree of cancer malignancy.
The present inventors discovered a novel matrix metalloproteinase (hereinafter referred to as xe2x80x9cMT-MMPxe2x80x9d.) and performed a structural analysis thereof.
As described hereafter, the present invention offers a novel metalloproteinase protein, DNA having a nucleotide sequence which encodes said protein, a plasmid having said DNA nucleotide sequence, a host cell harbouring said plasmid and monoclonal antibodies which specifically recognize the aforesaid metalloproteinase protein.
1. A native membrane-type matrix-metalloproteinase characterized by a continuous sequence of hydrophobic amino acids peculiar to membrane-binding proteins from amino acid number 533 to 562 in the C terminus domain shown in SEQ ID NO: 1,
2. A, native membrane-type matrix-metalloproteinase according to claim 1, characterized by the amino acid sequence from amino acid number 160 to 173, 320 to 333 and from 498 to 512 shown in SEQ ID NO: 1.
3. A native membrane-type matrix-metalloproteinase according to claim 1, characterized by the amino acid sequence from amino acid number 1 to 173, 320 to 333, 498 to 512 and 563 to 582 in SEQ ID NO: 1.
4. A DNA having the nucleotide sequence shown in SEQ ID NO: 2 which corresponds to the amino acid sequence of a membrane-type matrix-metalloproteinase according to claim 1, 2 or 3.
5. A plasmid containing a DNA having the nucleotide sequence according to claim 4 and expressing a membrane-type matrix-metalloproteinase according to claim 1, 2 or 3.
6. A host cell harbouring a plasmid containing a DNA having the nucleotide sequence according to claim 4, and expressing a membrane-type matrix-metalloproteinase according to claim 1, 2 or 3.
7. Monoclonal antibodies which peculiarly recognize a membrane-type matrix-metalloproteinase according to claim 1, 2 or 3.
8. A protein having the amino acid sequence shown in SEQ ID NO: 1.
9. A DNA having the nucleotide sequence shown in SEQ ID NO: 2 which encodes a protein having the amino acid sequence shown in SEQ ID NO: 1.
10. A plasmid containing a DNA having the nucleotide sequence shown in SEQ ID NO: 2, and expressing the protein shown in SEQ ID NO: 1.
11. A host cell harbouring a plasmid containing a DNA having the nucleotide sequence shown in SEQ ID NO: 2, and expressing the protein shown in SEQ ID NO: 1.
12. Monoclonal antibodies which peculiarly recognize a protein having the amino acid sequence shown in SEQ ID NO: 1.
The present invention is described in detail hereafter.
Using highly conserved sequences SEQ ID NOS: 3 and 4 selected from amino acid sequences of the known matrix metalloproteinase (MMP) family, the present inventors designed and synthesized an oligonucleotide primer having the sequences denoted by SEQ ID NOS: 5 and 6. A PCR was carried out using said oligonucleotide primer and a human placental cDNA library, the PCR products obtained were sequenced, and a 390 bp DNA fragment having a sequence non-homologous to known MMP was obtained. Using this 390 bp DNA fragment as a probe, the human placenta cDNA library was screened, and a cDNA in the positive phage clone obtained was sequenced. The nucleotide sequence is that denoted by SEQ ID NO: 2. A sequence identical to the nucleotide sequence in SEQ ID NO: 2 did not exist in the Genbank/EMBL DNA database, and DNA having this nucleotide sequence was ascertained to be completely novel.
The nucleotide sequence of the aforesaid cloned cDNA in SEQ ID NO: 2 had 3xe2x80x2 non-coding sequence and open reading frame that potentially encode 582 amino acid. An initiation codon was located at nucleotide number 112, and a stop codon was present at nucleotide number 1858. It was determined that this open reading frame encoded the 582 amino acid sequence in SEQ ID NO: 1, that a deduced signal sequence continued after the initiation codon, and that a hydrophobic domain SEQ ID NO: 7 specific to a membrane-binding protein of 20 or more linked hydrophobic amino acids was present from C-terminal amino acid number 533 to 562.
When homology between the amino acid sequence of MT-MMP and that of the known MMP family was analyzed, MT-MMP had high homology to the known MMP family, as shown in FIG. 2. The sequences best conserved in MT-MMP were active site sequences, as well as sequences proximal to processing site between precursor and mature substance conserved in the MMP family. The fact that MT-MMP has the structural characteristics of a membrane-binding protein, and the presence in MT-MMP of a sequence of linked hydrophobic amino acids (shown in Sequence Sheet sequence number 7) not found in the rest of the MMP family, strongly suggested that MT-MMP, unlike other MMP family, is a membrane-binding MMP.
When MT-MMP expression in various human tissues was studied by Northern Blot analysis with various tissue-derived Poly(A)RNA, high expression was seen in the placenta, lung and kidney (see FIG. 3). Likewise, results from Northern Blot analysis performed with RNA extracted from normal and tumour areas of human lung squamous cell carcinoma showed that MT-MMP is expressed peculiarly at tumour sites (see FIG. 4).
Finally, immunoprecipitation and immunostain experiments using anti-MT-MMP monoclonal antibodies showed that the MT-MMP pertaining to the present invention is expressed on a cell membrane without secretion of a gene product, and MMP-2 activation induced by the expression of MT-MMP was observed in the cells transfected with MT-MMP gene (Nature, 370:61-65, 1994).
Due to the achievements of the above-discussed research by the present inventors, the present invention offers a novel matrix metalloproteinase protein having the amino acid sequence in SEQ ID NO: 1.
In addition, the present invention offers DNA having the nucleotide sequence in SEQ ID NO: 2, which encodes a protein having the amino acid sequence in SEQ ID NO: 1; a plasmid containing and capable of expressing said DNA; and a host cell harbouring said plasmid. All host cells used in general recombinant DNA technology can be used as the aforementioned host cell, including prokaryotes such as E. coli and Baci lus subtilus; eukaryotes such as yeast, COS cells, CHO cells and 3T3 cells; and insect cells such as Sf21. Expression vectors corresponding to used host cells can be used as the aforementioned plasmid.
Furthermore, the present invention offers mRNA transcribed from DNA having the nucleotide sequence in SEQ ID NO: 2.
The present invention also offers a probe which hybridizes with the aforementioned DNA or RNA and specifically detects said DNA or RNA, and said probe may be one having any part of the nucleotide sequence in SEQ ID NO: 2, provided said probe is labeled by a generally used radioactive isotope or enzyme or the like, hybridizes specifically with said DNA or RNA in general blotting analysis and in situ hybridization, and accomplishes detection.
Furthermore, the present invention offers monoclonal and polyclonal antibodies which bind peculiarly with the MT-MMP pertaining to the present invention.
The monoclonal and polyclonal antibodies pertaining to the present invention can be prepared by a well-known method such as the method of Milstein et al. (Nature, 256:495-497, 1975) using human MT-MMP as an antigen. In this method, the antigen may be native human MT-MMP, recombinant human MT-MMP, or a synthetic peptide having a partial amino acid sequence of either.
By means of the present invention, DNA having a nucleotide sequence which encodes a protein with the amino acid sequence of the novel MT-MMP pertaining to the present invention can be cloned, and such DNA and a protein encoded by such DNA can be prepared by a genetic engineering technique. Through the use of a cDNA clone of such a novel MT-MMP, techniques generally used in genetic engineering can be used to clone the aforementioned nucleotide sequence into another vector or host. Based on the aforementioned cDNA nucleotice sequence, DNA appropriately suited to a probe may be designed and prepared. In addition, based on the nucleotide sequence of the MT-MMP pertaining to the present invention, techniques generally used in genetic engineering can be used to prepare a corresponding protein wherein appropriate mutation have been introduced into the MT-MMP amino acid sequence by substitution, deletion, insertion, displacement or addition of one or more amino acids. All such aforementioned derivatives may also be included in the present invention, provided that common metalloproteinase characteristics are conserved; namely, sequences proximal to processing site between precursor and mature substance, active site sequences and domain structure, and provided that the MT-MMP characteristic of a hydrophobic domain of linked hydrophobic amino acids present near the C terminus is conserved.
Use of the above-discussed various implementations of the present invention offers various technical means applicable to applications pertaining to diagnostic agents or diagnostic methods used for diagnosis of the presence of tumoar cells or for diagnosis of the degree of tumour malignancy, as well as applications in other medical or physiological fields.
The present invention is described in detail hereafter by means of Working Examples, but the present invention is not limited by these Working Examples.